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Cell Disruption

Various cell disruption techniques

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Cell Disruption

  1. 1. Cell Disruption
  2. 2.  Cell disruption is the process of obtaining intracellular fluid via methods that open the cell wall.  The overall goal in cell disruption is to obtain the intracellular fluid without disrupting any of its components  The method used may vary depending on the type of cell and its cell wall composition.  Irrespective of the method used, the main aim is that the disruption must be effective and the method should not be too harsh so that the product recovered remains in its active form.
  3. 3.  There are two types of cell disruption method which are following  Mechanical methods  Non Mechanical methods
  4. 4.  The method selected for large scale cell disruption will be different in every case, but will depend on:  Susceptibility of cells to disruption  Product stability  Ease of extraction from cell debris  Speed of method  Cost of method
  5. 5.  Mechanical methods are those methods which required some sort of force to separate out intracellular protein without adding chemical or enzyme  The main principle of the mechanical disruption methods is that the cells are being subjected to high stress via pressure, abrasion with rapid agitation with beads, or ultrasound.  Intensive cooling of the suspension after the treatment is required in order to remove the heat that was generated by the dissipation of the mechanical
  6. 6.  laboratory scale - high-pressure methods such as French press and Hughes press.  Industrial - the bead mill and high-pressure homogenizer 1. Bead mill 2. Ultrasonication 3. French press and high pressure homogeniser
  7. 7.  The main principle requires a jacketed grinding chamber with a rotating shaft, running in its center.  Agitators are fitted with the shaft, and provide kinetic energy to the small beads that are present in the chamber.  That makes the beads collide with each other.  The choice of bead size and weight is greatly
  8. 8.  The increased number of beads increases the degree of disruption, due to the increased bead-to-bead interaction.  The increased number of beads, however, also affects the heating and power consumption.  Glass, alumina, titanium carbide, zirconium oxide, zirconium silicate.  Glass beads with a diameter greater than 0.5 mm - yeast cells  diameter lesser than 0.5 mm - bacterial cells.
  9. 9.  The process variables are: agitator speed, proportion of the beads, beads size, cell suspension concentration, cell suspension flow rate, and agitator disc design.  Main issues related to bead mills are,  the high temperature rises with increase of bead volume,  poor scale-up,  high chance of contamination.
  10. 10.  First Order  k is a function of  Rate of agitation (1500- 2250 rpm)  Cell concentration (30- 60% wet solids)  Bead diameter (0.2 -1.0 mm)  Temperature
  11. 11.  Ultrasonic disruption is caused by ultrasonic vibrators that produce a high frequency sound with a wave density of about 20 kHz/s  A transducer then converts the waves into mechanical oscillations through a titanium probe, which is immersed into the cell suspension.  used for both bacterial and fungal cell disruption.  Bacterial cell - 30 to 60 sec, and yeast - 2 and 10min.  disadvantage: It’s very loud and has to be performed in an extra room
  12. 12. • Highly effective at lab scale (15-300W) • Poor at large scales • High energy requirements • Safety issues - noise • Heat transfer problems • Not continuous • Protein lability
  13. 13.  The cell suspension is drawn through a valve into a pump cylinder.  Then it is forced under pressure of up to 1500 bar, through a narrow annular gap and discharge valve, where the pressure drops to atmospheric.  Cell disruption is achieved due to the sudden drop in pressure upon the discharge, causing the cells to explode.  This method is one of the most widely known and used methods.  It is mostly used for yeast cells.
  14. 14.  protein release is dependent on several factors:  Temperature  intracellular location of the enzymes  number of passes  operating pressure.  biomass concentration.
  15. 15.  It consists of  High pressure positive displacement pump  Discharge valve with a restricted orifice  Cell suspension pumped through homogenizing valve at 200-1000 atm (depends on type of microorganism and concentration of cell suspension)  cell suspension is cooled as it exit the valve to reduce thermal denaturation of the product.
  16. 16.  By various stresses developed on the cell suspension  Stress is developed due to the impingement of high velocity jet of suspended cells on the stationary surface (impact ring)  The stress is expressed as dynamic pressure Ps - depends on jet velocity v, and fluid density ρ, Ps = ½ ρv2
  17. 17.  Normal and shear stresses are also generated  Normal stress – due to the fluid pass through the narrow channel of the orifice  Shear stress – rapid decrease in pressure due to cell suspension passes out of the orifice.
  18. 18.  Parameters which influence degree of cell disruption  Nature of microorganism – size, cell wall composition, thickness and concentration of microbial cell  Product location within the cell  Type of homogenizer –type of valve and seat  Operating pressure  Temperature  Noof passes of cell suspension through the homogenizer
  19. 19.  Cell disruption described by, first order kinetics  N – noof passes through valve  k – first order rate constant depends on operating pressure  P- operatting pressure  n – constant , varying with pressure, with pressure
  20. 20.  Non mechanical methods are further divided into three class which are following  Physical methods 1. Heat shock/ Thermolysis 2. Osmatic shock  Chemical methods  Enzymatic methods
  21. 21.  common in large scale production, easy, economical  Used if the products are stable to heat shock  It inactivates organism by disrupting the cell wall without affecting the products  The effect of heat shock depends on parameters such as pH ionic strength presence of chelating agents like EDTA which binds Mg presence of proteolytic and hydrolytic enzymes.
  22. 22.  Periplasmic proteins in G (-) bacteria are released when the cells are heated up to 50ºC.  Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.  Improved protein release has been obtained after short high temperature shocks, than when at longer temperature exposures at lower values.  The results are highly unreliable, as the protein solubility changes with temperature fluctuations.
  23. 23.  Caused by sudden change in salt concentration  Provided by dumping a given volume of cell into double volume of water.  The cells swell due to osmotic flow of water and then burst  Release the product into surrounding medium  Osmotic pressure, π, proportional to concentration of solutes and temperature, as given by van’t Hoff equation
  24. 24.  Susceptibility of cells to undergo disruption by osmotic shock depends on types of cells  Red blood cells easily disrupted  Animal cells after mincing or homogenizing the tissues  Plant cells most resistant  This technique is used if the product is in periplasmic region
  25. 25. 1. Alkali treatment 2. Detergent solubilization 3. Lipid solubilization by organic solvents 4. Enzymatic method
  26. 26.  Alkali treatment  Cheap and effective method but harsh  Alkali acts on the cellwall – saponification of lipids  pH 11-12, 20 -30 min  Proteases are inactivated by this method – it is used in the preparation of pyrogen free therapeutic enzymes
  27. 27.  Detergent solubilization  Addition of concentrated solution of detergent to about half the volume of cell suspension  Depends on pH and temperature  Detergents are capable of interacting both water and lipids  Detergent solubilize the lipids in the cellwall and form a micelle  In dilute solution detergents donot dissolve but in high concentration lipid solubilization begins suddenly and thereafter increases linearly with detergent concentration.
  28. 28.  The range of detergent concentration at which the abrupt changes in lipid solubility and surface tension of the medium occur is called critical micelle concentration and corresponds to the formation of micelle.  Anionic detergents – SDS, sodium sulphonate  Cationic detergents – CTAB  Non ionic detergent – triton X-100
  29. 29.  Cell wall permeabilization  Addition of organic solvents  Solvent is absorbed by cell wall resulting in swelling and ultimate rupture  Lower concentration – permeabilize the cell wall  This method is usefull in retaining the components of the cell for sequencial release of the desired product and use permeabilized cell as a porous bag  Toluene, benzene, xylenes, octanol
  30. 30.  to use digestive enzymes which will decompose the microbial cell wall  lysozyme is commonly used enzyme to digest cell wall of gram positive bacteria.  Lysozyme hydrolyzes β-1-4-glucosidic bonds in the peptidoglycan.  The cell wall of gram negative bacteria differs from the cell wall of gram positive bacteria so lysozyme is not very efficient in the case of gram negative cell wall.